Association of omega 3 polyunsaturated fatty acids with incident chronic kidney disease: pooled analysis of 19 cohorts

Abstract Objective To assess the prospective associations of circulating levels of omega 3 polyunsaturated fatty acid (n-3 PUFA) biomarkers (including plant derived α linolenic acid and seafood derived eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid) with incident chronic kidney disease (CKD). Design Pooled analysis. Data sources A consortium of 19 studies from 12 countries identified up to May 2020. Study selection Prospective studies with measured n-3 PUFA biomarker data and incident CKD based on estimated glomerular filtration rate. Data extraction and synthesis Each participating cohort conducted de novo analysis with prespecified and consistent exposures, outcomes, covariates, and models. The results were pooled across cohorts using inverse variance weighted meta-analysis. Main outcome measures Primary outcome of incident CKD was defined as new onset estimated glomerular filtration rate <60 mL/min/1.73 m2. In a sensitivity analysis, incident CKD was defined as new onset estimated glomerular filtration rate <60 mL/min/1.73 m2 and <75% of baseline rate. Results 25 570 participants were included in the primary outcome analysis and 4944 (19.3%) developed incident CKD during follow-up (weighted median 11.3 years). In multivariable adjusted models, higher levels of total seafood n-3 PUFAs were associated with a lower incident CKD risk (relative risk per interquintile range 0.92, 95% confidence interval 0.86 to 0.98; P=0.009, I2=9.9%). In categorical analyses, participants with total seafood n-3 PUFA level in the highest fifth had 13% lower risk of incident CKD compared with those in the lowest fifth (0.87, 0.80 to 0.96; P=0.005, I2=0.0%). Plant derived α linolenic acid levels were not associated with incident CKD (1.00, 0.94 to 1.06; P=0.94, I2=5.8%). Similar results were obtained in the sensitivity analysis. The association appeared consistent across subgroups by age (≥60 v <60 years), estimated glomerular filtration rate (60-89 v ≥90 mL/min/1.73 m2), hypertension, diabetes, and coronary heart disease at baseline. Conclusions Higher seafood derived n-3 PUFA levels were associated with lower risk of incident CKD, although this association was not found for plant derived n-3 PUFAs. These results support a favourable role for seafood derived n-3 PUFAs in preventing CKD.

NHLBI, with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS). Additional support was provided by R01AG023629 from the National Institute on Aging (NIA). A full list of principal CHS investigators and institutions can be found at CHS-NHLBI.org. The authors express their gratitude to the CHS participants.

EPIC-Norfolk: European Prospective Investigation into Cancer and Nutrition (EPIC) study (Norfolk)
General information: The EPIC-Norfolk study (DOI 10.22025/2019.10.105.00004) is a prospective study of 25,639 men and women aged 40-79 years in Norfolk, UK similar in characteristics to UK general population samples, who participated in a baseline survey in 1993-1997. 7,8 Among 25,639 adults, there were 7,384 adults with available data on fatty acid biomarkers measured in plasma phospholipid. Among them, eGFR at baseline and follow-up visits were available in 1,027 adults. Thus, the EPIC-Norfolk Study analysed 1,027 adults in this current study. For primary outcomes of incident chronic kidney disease, after excluding 101 adults with prevalent CKD at baseline, a total of 926 adults were eligible.

Measurement of fatty acid biomarker concentrations:
The original assays on 8,000 samples were carried out in the WHO International Agency for Cancer Research laboratories, Lyon, France. Because of laboratory constraints in Lyon, an additional 2,000 samples were analysed in Quotient Laboratories, UK, using the same methods and quality control standards. Citrated plasma straws were retrieved from liquid nitrogen storage, thawed at room temperature and 20 μg of di-palmitoyl-D31-phosphatidylcholine (Sigma, St. Louis, MO) internal standard was added to each 200 μL plasma sample. Following extraction of total lipids with chloroform/methanol, phospholipids were further purified by adsorption chromatography (LC-Si SPE, Supelco/Sigma, St. Louis, MO), transmethylated to fatty acid methyl esters and extracted with hexane. Analysis was carried out by gas chromatography with flame ionisation detection (220°C) using a 30 m x 0.32 mm x 0.2 µm SP2340 fused silica capillary column (Supelco/Sigma, St. Louis, MO). Carrier gas was Helium at a constant flow of 1.3 mL/min. Samples of 0.5 µl were introduced onto the column via on-column injection. The column was held initially for 1 min at 65°C, then programmed at 5°C/min to 135°C, then at 2°C/min to 200°C, and finally at 10°C/min to 220°C. Run time was 60 min.
Citrated plasma straws retrieved from storage were thawed, di-palmitoyl-D31-phosphatidylcholine Sigma) internal standard was added to each plasma sample, total lipids extracted, and purified by adsorption chromatography (LC-Si SPE, Supelco/Sigma). Plasma phospholipids were analysed by gas chromatography. Concentrations were measured by comparison of peak areas of individual FAs with the peak area of the internal standard using individual calibration curves.
Physical activity assessment: Physical activity was assessed at baseline using self-reported leisure and work physical activity questionnaire.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: Among 926 participants with available data on the fatty acid biomarkers and CKD incidence over the follow-up, 23 (2.5%) participants recorded missing information on one or more covariates needed in the most adjusted models. Missing information was imputed with chained equations based on regression models including all the relevant variables. Ten imputed datasets were generated, and a single imputed dataset was used for this project after confirming little variability in results from ten imputed datasets.
Funding support and acknowledgements: The EPIC-Norfolk study has received funding from the Medical Research Council (MR/N003284/1 MC-UU_12015/1 and MC_UU_00006/1) and Cancer Research UK (C864/A14136). FI, NGF, and NJW were funded by the United Kingdom Medical Research Council Epidemiology Unit core grant (MC_UU_00006/1 and MC_UU_00006/3); NGF and NJW acknowledge National Institute for Health Research (NIHR) Biomedical Research Centre Cambridge (IS-BRC1215-20014). NGF is an NIHR Senior Investigator. The authors express their gratitude to the EPIC-Norfolk participants.

EPIC-Potsdam: European Prospective Investigation into Cancer and Nutrition (EPIC) study (Potsdam)
General information: The EPIC-Potsdam study is part of the multi-centre prospective cohort study EPIC. 9 In Potsdam, Germany, 27,548 subjects (16,644 women aged mainly 35-65 years and 10,904 men aged mainly 40-65 years) from the general population were recruited between 1994 and 1998. A case-cohort study within the prospective EPIC-Potsdam study was designed. We randomly selected 2,500 individuals from all participants of the EPIC-Potsdam study population who provided blood samples (n=26,444) for a subcohort. Cases comprised all incident type 2 diabetes cases, which were diagnosed until 2009 (n=820).
From the initial case-cohort, 371 participants came for the follow-up visit (EPIC-DZD study) into the study centre and of these, 259 participants had available creatinine measurements from baseline and follow-up visits and fatty acid biomarker measurements from baseline, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 6 adults with prevalent CKD at baseline, a total of 253 adults were eligible for the current analysis.
Measurement of fatty acid biomarker concentrations: Thirty milliliters of blood were taken from each participant during baseline examination and were centrifuged at 1,000 g for 10 min at 4°C. Plasma, serum, red blood cells, and buffy coat were removed and stored at -80°C. The erythrocyte membrane fatty acids were analysed at the Laboratory of the Dutch National Institute for Public Health and Environment between February and June 2008. Briefly, fatty acid methyl esters (FAME) were separated on a GC-3900 gas chromatograph (Varian Inc., Middelburg, Netherlands) equipped with a 100 m x 0.25 mm ID WCOT-fused silica capillary column and flame ionisation detector with separation of FAME peaks based on mixed FAME standards (Sigma Aldrich, St Louis, USA). The Galaxie software version 1.9.3.2 (Varian Inc.) was used for quantification and identification of peaks. The FAs were expressed as the percentage of total FAs present in the chromatogram. Intraassay CVs (%): ALA: 10.2, EPA: 3.1, DPA: 2.2, DHA: 2.4 Physical activity assessment: Physical activity was assessed via self-reports made in a computer-guided interview. We considered sport activities and biking as leisure time physical activities, both calculated as the average time spent per week during the twelve months before baseline recruitment.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: There was only one missing data in systolic blood pressure, which were excluded from the analysis.
Funding support and acknowledgements: The EPIC-Potsdam study was supported by the German Federal Ministry of Science (01 EA 9401) and the European Union (SOC 95201408 05F02) for the recruitment phase. The follow-up of the EPIC-Potsdam Study was supported by the German Cancer Aid (70-2488-Ha I) and the European Community (SOC 98200769 05F02). This work was furthermore supported by a grant from the German Ministry of Education and Research (BMBF) and the State of Brandenburg (DZD grant 82DZD00302). The present analysis was also supported by a grant from the German Research Foundation (DFG, SCHU 1516/5-1). The authors thank the Human Study Centre (HSC) of the German Institute of Human Nutrition Potsdam-Rehbrücke, namely the trustee and the data hub for the processing, and the participants for the provision of the data, the biobank for the processing of the biological samples and the head of the HSC, Manuela Bergmann, for the contribution to the study design and leading the underlying processes of data generation. The authors thank Hans Cremers for the excellent performance of the FA analysis.

FDPS: Finnish Diabetes Prevention Study
General information: The Finnish DPS is a randomised study aimed at preventing type 2 diabetes through intensive lifestyle intervention, carried out at five study clinics in Finland. 10,11 The randomised trial started in November 1993, the recruitment period lasted until June 1998 and the intervention period lasted until the end of 2001. The last follow-up data were collected in 2009 with a follow-up over 13 years from baseline. Participants were recruited at five field centers (Kuopio, Helsinki, Tampere, Turku and Oulu). The cohort consisted of 522 non-institutionalised men and women. Originally, subjects at baseline were all with impaired glucose tolerance defined by repeated oral glucose tolerance tests (OGTT) (mean value of 2-hour plasma glucose concentrations 7.8-11.0 mmol/L in two OGTTs). For the fatty acid measurements, only subjects who were non-diabetic by the revised WHO 1999 criteria at baseline and had stored serum samples available from the active study period (n=407).
There were 388 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 33 adults with prevalent CKD at baseline, a total of 355 adults were eligible for the current analysis.

Measurement of fatty acid biomarker concentrations:
The total serum fatty acid composition was measured by TETHYS Bioscience Inc. (Emeryville, CA) in 2010, using stored (-80 o C) serum samples taken during the active intervention period. 12 The lipids from serum were extracted in the presence of authentic internal standards by using chloroform:methanol (2:1 v/v). 6 The total lipid extract was trans esterified in 1% sulfuric acid in methanol in a sealed vial under a nitrogen atmosphere at 100°C for 45 min. The resulting extract was neutralised with 6% potassium carbonate and the fatty acid methyl esters were extracted with hexane. The isolated fatty acid methyl ester extract was then prepared for gas chromatography by sealing the hexane extracts under nitrogen. The fatty acid methyl esters were separated and quantified by capillary gas chromatography (Agilent Technologies model 6890) equipped with a 30 m DB 88MS capillary column (Agilent Technologies) and a flame ionisation detector. Proportions of fatty acids are expressed as molar percentages (mol/mol of all fatty acids). The intra-and inter-assay CV% for individual fatty acids were ≤10% and ≤12%, respectively.
Physical activity assessment: Physical activity was assessed via self-reported questionnaires.

Statistical methods for incident CKD analysis: Cox regression
Missing data in covariates: Participants with missing data in covariates were excluded from the analysis (<5%).
Funding support and acknowledgements: The FDPS study has been financially supported by the Academy of Finland (128315, 129330), Ministry of Education, Novo Nordisk Foundation, Yrjö Jahnsson Foundation, Juho Vainio Foundation, Finnish Diabetes Research Foundation, Finnish Foundation for Cardiovascular Research, Unilever, and Competitive Research Funding from Tampere, Kuopio and Oulu University Hospitals. The study sponsors had no role in the design and conduct of the study; the collection, analysis and interpretation of the data; or the preparation, review or approval of the manuscript. The authors express their gratitude to the FDPS participants.

FHS: Framingham Heart Study
General information: Our analysis focused on the Framingham Heart Study (FHS) Offspring sample, a population based longitudinal study of families living in Framingham, Massachusetts. 13 The offspring study was initiated in 1971 and consisted of a sample of 5,124 individuals, offspring of the original cohort and their spouses.
The offspring started with 5,124 individuals in 19715,124 individuals in . Exam 8 (in 2008 consisted of 3,021 individuals, having lost 2,003 individuals due to death and loss to follow-up. Fatty acid data was available for 2,692 adults of the 3,021. Of these 2,692 individuals, there were 2,041 adults with available data on eGFR at baseline (Exam 8) and follow-up (Exam 9) visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 146 adults with prevalent CKD at baseline, a total of 1,895 adults were eligible for the current analysis.

Measurement of fatty acid biomarker concentrations:
The fatty acid composition of red blood cell (RBC) samples was analysed by gas chromatography equipped with a SP 2560 capillary column after direct transesterification for 10 minutes in boron trifluoride/ methanol and hexane at 100 o C as previously described. This technique generates fatty acids primarily from RBC glycerophospholipids. RBCs were isolated from blood drawn after a 10-12 h fast and frozen at -80 °C immediately after collection. All fatty acids present at >1% abundance had CVs of ≤7%.
Physical activity assessment: Self-reported values on typical activities and rest hours per day were gathered as part of the Physical Activity Questionnaire. Metabolic equivalents per week were computed using standard scoring approaches.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: Participants with missing data in covariates were excluded from the analysis (<5%).
Funding support and acknowledgements: The FHS study is conducted and supported by the National Heart, Lung and Blood Institute (NHLBI) and in collaboration with Boston University (Contract No. N01-HC-25195). The authors express their gratitude to the FHS participants.

Hisayama: Hisayama Study
General information: The Hisayama Study is an ongoing, population-based prospective cohort study of cardiovascular disease and its risk factors in the town of Hisayama, a suburb in the metropolitan in Japan. 14 A total of 3,293 residents who were aged 40 years or older, and had no missing values for serum fatty acid levels were enrolled in the present study. There were 2,934 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 221 adults with prevalent CKD at baseline, a total of 2,713 adults were eligible for the current analysis.
Measurement of fatty acid biomarker concentrations: Serum fatty acids levels were assayed by gas chromatography (SRL, Tokyo, Japan). Briefly, total lipids in plasma were extracted according to the Folch's procedure 6 , followed by hydrolysis to free fatty acids. Free fatty acids were esterified with potassium methoxide/methanol and boron trifluorideemethanol. The methylated fatty acids were analysed using GC-17A gas chromatograph (Shimadzu Corporation, Kyoto, Japan) with omegawax-250 capillary column (SUPELCO, Sigmae Aldrich Japan, Tokyo, Japan). Reproducibility (i.e. the coefficient of variation) of the determination of serum EPA, DHA, and ALA levels by this method was reported to be 4.4%, 2.3%, and 3.8%, respectively. 15 Physical activity assessment: Physical activity was assessed as frequency of leisure-time exercise/week.

Statistical methods for incident CKD analysis: Cox regression
Missing data in covariates: There were missing data on alcohol consumption (2.9%) and urine albumin-creatinine ratio (0.8%). For missing covariate data, dummy variable indicating missing value was used.
Funding support and acknowledgements: This study was supported in part by Grants-in-Aid for Scientific Research A (JP16H02692), B (JP17H04126, JP18H02737,and JP19H03863), C (JP18K07565, JP18K09412, JP19K07890, JP20K10503,and JP20K11020), Early-Career Scientists (JP18K17925) , and Research Activity Start-up (JP19K23971) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by Health and Labour Sciences Research Grants of the Ministry of Health, Labour and Welfare of Japan (20FA1002); and by the Japan Agency for Medical Research and Development (JP20dk0207025, JP20km0405202, JP20fk0108075). The authors express their gratitude to the Hisayama residents and the staff of the Division of Health and Welfare of Hisayama for their cooperation in this study.

InCHIANTI: Invecchiare in Chianti
General information: The InCHIANTI study is a population-based epidemiological study aimed at evaluating the factors that influence mobility in the older population living in the Chianti region in Tuscany, Italy. 16 1,616 residents were selected from the population registry of Greve in Chianti (a rural area: 11,709 residents with 19.3% of the population greater than 65 years of age), and Bagno a Ripoli (Antella village near Florence; 4,704 inhabitants, with 20.3% greater than 65 years of age). The participation rate was 90% (n=1,453), and the subjects ranged between 21-102 years of age. Following the baseline visit between 1998-2000, there were four follow up visits at 2001-2003, 2004-2006, 2007-2009, and 2013-2014. Among 1,453 adults, there was 1,177 adults with available data on fatty acid biomarkers and eGFR at baseline. After excluding 80 adults (6.8%) with missing data in the covariates, there were 962 participants with data on follow-up eGFR, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 132 adults with prevalent CKD at baseline, a total of 830 adults were eligible for the current analysis.
Measurement of fatty acid biomarker concentrations: Plasma fatty acids measurement method has been described previously. 17 Briefly, blood samples were collected in the morning after a 12-hr overnight fast. Aliquots of plasma were immediately obtained and stored at -80 o C. Fatty acid methyl esters (FAME) were prepared through transesterification using Lepage and Roy's method with modification. 6,18 Separation of FAME was carried out on an HP-6890 gas chromatograph (Hewlett-Packard, Palo Alto, CA) with a 30-m fused silica column (HP-225; Hewlett-Packard). FAMEs were identified by comparison with pure standards (NU Chek Prep, Inc., Elysian, MA). For quantitative analysis of fatty acids as methyl esters, calibration curves for FAME (ranging from C14:0 to C24:1) were prepared by adding six increasing amounts of individual FAME standards to the same amount of internal standard (C17:0; 50xg). The correlation coefficients for the calibration curves of fatty acids were in all cases higher than 0.998 in the range of concentrations studied. Fatty acid concentration was expressed as a percentage of total fatty acids. The coefficient of variation for all fatty acids was on average 1.6% for intraassay and 3.3% for inter-assay.
Physical activity assessment: Physical activity in the previous year was assessed as i) sedentary (hardly any physical activity, ii) mostly sitting/some walking, iii) Light exercise 2-4 hr/week, iv) moderate 1-2 hr/week, v) moderate exercise >3 hr/week, vi) intensive exercise many times/week and vii) walk over 5 km/day for 5 days for at least 5 years.

Statistical methods for incident CKD analysis: Cox regression
Missing data in covariates: As described in the "General information" section above, participants with missing data in covariates were already excluded from the analysis.

KIHD: Kuopio Ischaemic Heart Disease Risk Factor Study
General information: The KIHD study was designed to investigate risk factors for cardiovascular diseases, atherosclerosis, and related outcomes in a population-based, randomly selected sample of men from eastern Finland. 19 The baseline examinations were carried out in [1984][1985][1986][1987][1988][1989]. A total of 2,682 men who were 42, 48, 54 or 60 years old at baseline (82.9% of those eligible) participated. The 20-year examinations were conducted in 2005-2008 for 1,241 men (79.7% of the eligible).
Among 2,682 men, there was 1,091 men with available data on fatty acid biomarkers and eGFR at baseline and followup visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 9 men with prevalent CKD at baseline, a total of 1,082 men were eligible for the analysis.
Measurement of fatty acid biomarker concentrations: Venous blood samples were collected between 8AM and 10AM after an overnight fast. Serum total fatty acids were determined from frozen samples with a NB-351 capillary column (HNU-Nordion, Helsinki, Finland) by a Hewlett-Packard 5890 Series II gas chromatograph (Hewlett-Packard Company, Avondale, PA, USA, since 1999 Agilent Technologies Inc., USA) with a flame ionisation detector. Serum was extracted with chloroform-methanol and fatty acids were methylated with methanol and sulphuric acid prior to gas chromatography. Each analyte had an individual reference standard and the analytes were quantified with an internal standard method using eicosane. The coefficient of variation (CV%) for repeated measurements of fatty acids was 12.8% for ALA, 9.4% for EPA, 12.7% for DPA, and 11.9% for DHA.
Physical activity assessment: Physical activity was evaluated based on the 12-month leisure-time physical activity questionnaire and expressed as kcal/day.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: The cohort mean was used to replace missing values in covariates (n=4).
Funding support and acknowledgements: The KIHD study was supported mainly by the funding from the Academy of Finland to Jukka T. Salonen. The authors express their gratitude to the KIHD participants.

MAS: Memory and Ageing Study
General information: The Sydney Memory and Ageing Study (MAS) is a longitudinal study which began in 2005 with the aims of investigating rates and predictors of healthy cognitive ageing, mild cognitive impairment (MCI) and dementia in older Australians. 20 Over the last 14 years, MAS has collected biomarker, genetic/epigenomic, neuroimaging, cognitive, proteomics/lipidomics, health, and lifestyle data to determine what factors are associated with cognitively normal ageing and progression to MCI or dementia. At baseline, there were 1,037 older adults without dementia, aged 70-90 recruited from Eastern Sydney, Australia. Among 1,037 adults, there were 395 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits without any missing data on covariates, who were included in the analysis for both primary and secondary outcomes as there were no participants with prevalent CKD at baseline.

Measurement of fatty acid biomarker concentrations:
Twenty-nine free, bound and total fatty acids in plasma was quantified using negative ion chemical ionisation gas chromatography -mass spectrometry (GCMS) approach as originally reported by Quehenberger O et al who developed a rapid protocol for the extraction of free and bound fatty acids from complex biological samples including plasma. 21 Fatty acids were quantified using the stable isotope dilution method, which was based on the principle that each target fatty acid was compared to a deuterated analog with similar chemical and structural properties which was added to all samples and standards at identical amounts. This allowed accurate quantification and also compensated for any losses during sample preparation and analysis. Enrichment and extraction of free fatty acids were achieved through addition of acidified methanol and isooctane and then deuterated fatty acids which acted as internal standards for quantification and to compensate for any losses during sample preparation. Samples were shaken and the two resulting solvent phases separated. The upper isooctane layer containing the free fatty acid fraction was removed, and extraction steps repeated and resulting extractions pooled. Samples were then evaporated to dryness. Hydrolysis of the remaining methanol fraction containing the esterified or bound fatty acid fraction was completed using KOH and incubation for 1 hr at 37 o C, followed by extraction of the released fatty acid using isooctane procedure described above. All samples were then derivatised to pentafluorobenzyl esters and reconstituted in isooctane before analysis. Standard curves of primary (unlabelled) fatty acid standards with added internal standards were run alongside samples. The ratios of unlabelled to labelled standards from the standard curves were used to quantify levels of fatty acids in plasma samples. Total fatty acid levels were calculated as sum of free and bound fatty acids, and then % of individual fatty acids of interest of total values were calculated for ALA, DHA, DPA and EPA. Intra-assay CVs were generally <15% and inter-assay CVs <5% for all fatty acids tested.
Physical activity assessment: The physical activity index represented the number of physical activities participated in, regardless of the amount or intensity of participation. The maximum score was 10 points. Data was categorised into three groups.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: There were 20 (5.1%) participants with missing data on the use of any anti-hypertensive drugs and use of lipid-lowering drugs. There was no missing data for other covariates. Imputation was performed for missing data.
Funding support and acknowledgements: The MAS study has been funded by three National Health & Medical Research Council (NHMRC) Program Grants (ID No. ID350833, ID568969, and APP1093083). The fatty acid analysis was funded by the NHMRC Investigator Grant (APP1196150). The authors thank the participants and their informants for their time and generosity in contributing to this research. The authors also acknowledge the MAS research team: https://cheba.unsw.edu.au/research-projects/sydney-memory-and-ageing-study. Blood samples were collected by South Eastern Area Laboratory Service (SEALS). The authors express their gratitude to the MAS participants.

MESA: Multi-Ethnic Study of Atherosclerosis
General information: The Multi-Ethnic Study of Atherosclerosis (MESA) is a study of the characteristics of subclinical cardiovascular disease (disease detected non-invasively before it has produced clinical signs and symptoms) and the risk factors that predict progression to clinically overt cardiovascular disease or progression of the subclinical disease. 22 MESA researchers study a diverse, population-based sample of 6,814 asymptomatic individuals of European, African, Hispanic, and Chinese American ancestry ascertained across six field centres across the United States. Baseline data for the current analyses were taken from the first clinic exam conducted in 2000-2002. In addition to yearly phone calls, follow-up clinic exams are conducted approximately every two years, and at the time the current analyses were conducted incident diabetes was available until the fifth clinic exam conducted in 2010-2012. Among 6,814 adults, there was 1,841 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 159 adults with prevalent CKD at baseline, a total of 1682 adults were eligible for the current analysis.
Measurement of fatty acid biomarker concentrations: Fatty Acid analysis was performed on plasma, that had been stored at -70 o C, using the method previously described. 23 For the extraction of phospholipid fatty acids, plasma was diluted in saline and lipids were extracted from with a mixture of chloroform:methanol, and cholesterol, triglycerides and phospholipid subclasses were separated on a silica thin-layer chromatography plate in a solvent mixture of petroleum ether, diethyl ether, and glacial acetic acid. The band of phospholipids is harvested for the formation of methyl esters. Fatty acid methyl esters are prepared with 14% boron trifluoride in methanol, incubated at 80ºC for 90 minutes, and extracted with petroleum ether. The final product is dissolved in heptane and injected onto a capillary Varian CP7420 100-m column with a Hewlett Packard 5890 gas chromatograph equipped with a HP6890A autosampler. The gas chromatograph is configured for a single capillary column with a flame ionisation detector and interfaced with HP ChemStation software. Separation of individual fatty acids was obtained over an 80-minute run. Fatty acids are expressed as a percent of total fatty acids. The following CVs were obtained on 20 blind duplicates: ALA= 2.4%; EPA= 3.3%; DPA = 2.9%, DHA= 2.7%.
Physical activity assessment: The MESA Typical Week Physical Activity Survey (TWPAS) was adapted from the Cross-Cultural Activity Participation Study and designed to measure weekly duration and frequency of various leisure physical activities. The survey has 28 items in categories of household chores, lawn/yard/garden/farm, care of children/adults, transportation, leisure walking, dancing and sport activities, conditioning activities, leisure activities, and occupational and volunteer activities. Questions differentiated between light-, moderate-, and heavy-intensity activities. Minutes of activity were summed for each activity and multiplied by metabolic equivalent (MET) level.

Statistical methods for incident CKD analysis: Cox regression
Missing data in covariates: Participants with missing data in covariates were excluded from the analysis.

METSIM: METabolic Syndrome in Men
General information: The METSIM study includes 10,197 men, aged from 45 to 73 years at entry, randomly selected from the population register of the Kuopio town, Eastern Finland, and examined in 2005-2010 (baseline). [24][25][26] The aim of the study is to investigate nongenetic and genetic factors associated with type 2 diabetes and cardiovascular disease, and with cardiovascular risk factors in both cross-sectional and longitudinal settings.
Fatty acid composition of erythrocyte membranes and plasma lipids was available in 1,364 participants at baseline. There were 1,158 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 6 adults with prevalent CKD at baseline, a total of 1,152 adults were eligible for the current analysis.

Measurement of fatty acid biomarker concentrations:
Phospholipids, cholesterol esters and triglycerides: Lipids were extracted from plasma sample with chloroformmethanol (2:1) and lipid fractions were separated with an aminopropyl column. FAs in lipid fractions were transmethylated with 14% borontrifluoride in methanol. Finally, fatty acid methyl esters were analysed by 7890A gaschromatograph (Agilent Technologies, Inc., Wilmington, DE, USA) equipped with a 25-m free FA phase column (Agilent Technologies). Cholesteryl nonadecanoate (Nu Chek Prep, Inc., Elysian, MA, USA), trinonadecanoin and phosphatidylcholine dinonadecanoyl (Larodan Fine Chemicals, Malmo, Sweden) served as internal standards. The intra-assay CVs for the n-3 fatty acids were between 0.3-3.6% and the inter-assay CVs between 2.0-14.0% Erythrocyte membranes: Erythrocytes were separated from EDTA-blood and then hemolysed in the tris-HCl buffer (pH 7.6, 10 mmol/L). Fatty acid methyl esters were prepared by direct trans-esterification using acetyl chloride and analysed by 7890A gas-chromatograph (Agilent Technologies, Inc., Wilmington, DE, USA) equipped with a 25-m free FA phase column (Agilent Technologies). Pure standards (NU Chek Prep Inc) were used to identify FA methyl esters and to prepare calibration curves. Heptadecanoic acid methyl ester (17:0) served as an internal standard. The intra-assay CVs for the n-3 fatty acids were between 1.1-3.3% and the inter-assay CVs between 4.2-9.9% Physical activity assessment: Leisure time physical activity was assessed by self-report at baseline using 4 categories: 1st=a little or none 2nd= Physical exercise in context of other hobbies or physical exercise occasionally 3rd= Physical exercise ≤2 times a week at least 30 min at a time 4th= Physical exercise regularly ≥3 times a week at least 30 min at a time

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: There was only one missing value (0.1%) in prevalent diabetes, which was excluded from the analysis.
Funding support and acknowledgements: The METSIM Study was funded by the grants from The European Union, the Academy of Finland, and the Juselius Foundation. The authors express their gratitude to the METSIM participants.

NHAPC: Nutrition and Health of Aging Populations in China
General information: The NHAPC study is a prospective population-based cohort study of people 50-70 years old in Beijing and Shanghai at baseline initiated to evaluate risk factors for metabolic disease. [27][28][29] Participants were recruited at two large cities representing the north and the south of China, respectively, two urban districts and one rural district in each city (Dongcheng, Beijing; Xuanwu, Beijing; Changping, Beijing; Luwan, Shanghai; Zhabei, Shanghai; Fengxian, Shanghai) from random samples of Medicare eligibility lists. The cohort consists of 3,289 noninstitutionalised men and women, recruited in 2005, 2,529 participants were successfully followed for 6 years.
Among 3,289 adults, there were 2,240 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits. After excluding 100 adults with outliers in any of the fatty acids (defined as < 25 th percentile -(1.5 x interquartile range) or >75 th percentile + (1.5 x interquartile range)), 2,140 adults were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, 94 adults with prevalent CKD at baseline were excluded, a total of 2,046 adults were eligible for the current analysis.
Measurement of fatty acid biomarker concentrations: Erythrocyte membrane fatty acids were measured at the Mass Spectrometry Subplatform in the Public Technical Service Center of Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences (Shanghai, China) using stored blood samples in 2005. Total lipids were extracted from plasma using the methods of Folch et al. 6 Erythrocyte samples were first extracted by hexane and isopropanol and then trans-methylated with methanol and sulfuric acid. Fatty acid methylesters were subsequently analysed by gas chromatography-cationic flame system (Agilent 6890GC, Bellefonte, Pennsylvania); The chromatographic column is a fused quartz capillary column SP2560(Supelco, Belefonte, Pennsylvania). According to the internal standard and known mass of fatty acid methyl ester mixture (GLC569B; NuCheck Prep, Elysium, Minnesota) as the standard to quantification. All fatty acids were processed at the Laboratory in Shanghai Institute of Nutrition and Health (Shanghai, China). For this analysis, the relative content of each fatty acid is expressed as the ratio of its area under the peak to the area under the peak of the total fatty acid. The CVs for de novo fatty acid assays ranged from 0.34% for 18:1n29 to 9.8% for 16:1n27. Intraassay coefficients of variation (CV; percentages) were 2.7% for ALA, 25.8% for EPA, 3.6% for DPA, 3.4% for DHA, and 0.7% for linoleic acid. The corresponding inter-assay CV were 7.8, 9.3, 14.1, 10.0, and 7.7%, respectively, for these fatty acids.
Physical activity assessment: Physical activity levels were assessed using a shortened version of the International Physical Activity Questionnaire, which collected the frequency and average duration of physical activity such as heavy, medium, light and sedentary activities over the past 7 days.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: There was no missing data for covariates.
Funding support and acknowledgements: The NHAPC study was funded by grants KSCX2-2-25, 04DZ14007, 200306 and 973 Program 2006CB503900 from Chinese Academy of Sciences, from the Science and Technology Commission of Shanghai Municipality, from the Shanghai-Unilever Research Development Fund and the Ministry and Technology of China, respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors express their gratitude to the NHAPC participants.

PIVUS: Prospective Investigation of the Vasculature in Uppsala Seniors
General information: The PIVUS study started in 2001 with the primary aim to investigate the predictive power of different measurements of endothelial function and arterial compliance in a random sample of just over 1,000 subjects aged 70 living in the community of Uppsala. 30 As secondary aims, the study also included measurements of cardiac function and structure by ultrasound and magnetic resonance imaging, evaluation of atherosclerosis by ultrasound and magnetic resonance imaging, 7-day food intake recordings, detailed electrocardiogram analysis, cardiovascular autonomic function, body composition by DXA, DNA analysis and lung function, as well as a number of biochemical markers.
There were up to 1,016 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 123 adults with prevalent CKD at baseline, a total of 893 adults were eligible for the current analysis.
Measurement of fatty acid biomarker concentrations: Fatty acid composition in cholesterol esters and phospholipids were measured by gas chromatography. Serum (0.5 mL) was mixed with 2.5 mL methanol, 5 mL chloroform (with 0.005% added butylated hydroxytoluene, BHT) and 7.5 mL NaH2PO4 (0.2 mol/L) and stored in 4°C over night for lipid extraction. The chloroform phase was then removed with a syringe and evaporated to dryness on a 30°C heating block using nitrogen gas. The lipid residue was dissolved in chloroform and the lipid fractions were separated by thin -layer chromatography (TLC); the adsorbent containing POPOP as fluorescent agent. The TLC-plates were eluted at room temperature with the solvent system petroleum ether/diethyl ether/acetic acid (81:18:1 by volume). The lipid fractions were visualised in UV light and the spots containing cholesterol esters and phospholipids were scraped off into vials and methylated at 60°C overnight after addition of 2 mL H2SO4 (5%) in methanol. The fatty acid methyl esters were extracted into 3 mL petroleum ether (0.005% BHT) after addition of 1.5 mL distilled water. The phases were separated after thorough mixing and centrifugation at 1,500g for 10 min. The petroleum ether phase was pipetted off and the solvent was evaporated under nitrogen gas on a 30°C heating block. The fatty acid methyl esters were dissolved in 120 µL hexane and placed in vials. The fatty acid methyl esters were separated by gas-liquid chromatography on a 30-m glass capillary column coated with Thermo TR-FAME (Thermo Electron Corporation, USA) with helium gas as a carrier gas. An Agilent Technologies system consisting of model GLC 6890N, autosampler 7683 and Agilent ChemStation was used. The temperature was programmed to 150-260°C. The fatty acids were identified by comparing each peak´s retention time with fatty acid methyl ester standards Nu Check Prep (Elysian, MN, USA). Fatty acids are presented as the relative sum of the fatty acids analysed in each compartment. For concentrations below detection limit, proportions were imputed as a random value between 0 and the minimum quantified proportion in the cohort.
Physical activity assessment: Physical activity levels were assessed using self-reported activities.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: Participants with missing data in covariates were excluded from the analysis (12%).
Funding support and acknowledgements: The PIVUS study was supported by Uppsala University Hospital; The Swedish Research Council for Health, Working Life and Welfare; The Swedish Research Council (K2015-54X-22081-04-3); and The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (2016-01639). The authors express their gratitude to the PIVUS participants.

ULSAM: Uppsala Longitudinal Study of Adult Men
General information: The ULSAM study is an ongoing, longitudinal, epidemiologic study based on all available men, born between 1920 and 1924 and living in Uppsala County, Sweden. 31 The men were investigated at the ages of 50, 60, 70, 77, 82, 88 and 93 years. The reinvestigations in ULSAM were based on the previous investigations. Full screening and official registry data are available in our databases and more data is continuously added. Among 2,294 adults, there were up to 1,055 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 0 adults with prevalent CKD at baseline, a total of up to 1,055 adults were eligible for the current analysis.

Measurement of fatty acid biomarker concentrations:
For analysis of the fatty acid composition of the serum cholesterol esters, serum was extracted with a hexane-isopropanol solution (1+4). Cholesterol esters were separated from the extract by thin layer chromatography before inter-esterification (acidic methanol at 85°C, 2 h), and free cholesterol liberated in the reaction was removed by an aluminium oxide column to avoid contamination of the gas liquid chromatography column. The percentage composition of methylated fatty acids 14:0 to 22:6 was determined by gas chromatography (a 25 m NB-351 silica capillary column, i.d. 0.32 mm, phase layer 0.20 mm) with use of a flame ionisation detector and with helium as carrier gas. Every 25th sample was a serum control pool. The precision of the between-series analysis (n=35) varied from 2% (large peaks) to 10% (smaller peaks) and between successive gas chromatography runs (n=17) from 0.2 to 5% (CV).
Physical activity assessment: Physical activity was assessed by self-reported questionnaire, and participants were classified into sedentary, moderate, strenuous and athletic categories according to the duration and vigorousness of their physical activity.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: Participants with missing data in covariates were excluded from the analysis (14%).

WHIMS: Women's Health Initiative Memory Study
General information: WHIMS was a randomised trial which examined the effects of postmenopausal hormone therapy on cognitive function in women aged 65-80 years. 32,33 Recruitment began in 1995.
Among 7,479 adults, there was 1,785 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 99 adults with prevalent CKD at baseline, a total of 1,686 adults were eligible for the current analysis.

Measurement of fatty acid biomarker concentrations:
The fatty acid composition of red blood cell (RBC) samples was analysed by gas chromatography equipped with a SP 2560 capillary column after direct transesterification for 10 minutes in boron trifluoride/ methanol and hexane at 100 o C as previously described. 34 This technique generates fatty acids primarily from RBC glycerophospholipids. During the aliquoting phase, the RBC samples were stored improperly at -20°C for a period of approximately 2 weeks, causing oxidative degeneration of the PUFAs before measurement. The original FA levels were estimated with multiple imputations using independent data on fatty acid degradation and length of time the samples were exposed to -20°C. 34 All fatty acids present at >1% abundance had CVs of ≤6.5%.
Physical activity assessment: Self-reported information on sedentary behaviour and usual physical activity was assessed as Mets/week.

Statistical methods for incident CKD analysis: Poisson regression
Missing data in covariates: Participants with missing data in covariates were excluded from the analysis (<5%).
Funding support and acknowledgements: The WHI program is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, U.S. Department of Health and Human Services through contracts HHSN268201600018C, HHSN268201600001C, HHSN268201600002C, HHSN268201600003C, and HHSN268201600004C. The authors thank the WHI investigators and staff for their dedication, and the study participants for making the program possible. A listing of WHI investigators can be found at http://www.whi.org/researchers/Documents%20%20Write%20a%20Paper/WHI%20Investigator%20Short%20List.pdf.

3C Study: Three City Study (3C)
General information: The Three-City (3C) study is an ongoing multi-centre prospective cohort study of vascular risk factors for dementia which started in 1999-2000 and included 9,294 community dwellers in three French cities: Bordeaux (n=2,104), Dijon (n=4,931) and Montpellier (n=2,259). 35 Individuals living in one of these cities, aged 65 years and over and not institutionalised were eligible for recruitment into the 3C study. The protocol of the 3C study has been approved by the Consultative Committee for the Protection of Persons participating in Biomedical Research of the Kremlin-Bicêtre University Hospital (Paris). All participants gave their written informed consent. The baseline data collection included socio-demographic and lifestyle characteristics, symptoms and complaints, main chronic conditions, medication use, neuropsychological testing, clinical examination including blood pressure measurement, electrocardiogram and blood sampling. Total plasma fatty acids were measured at baseline from fasting blood samples among 1,416 individuals from the Bordeaux centre. Moreover, fatty acid composition of red blood cell membrane phospholipids were measured at baseline from fasting blood samples among 670 individuals from the Bordeaux and Montpellier centres. Among 2,104 adults from 3C Bordeaux study centre, there was 1,130 adults with available data on plasma fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 111 adults with prevalent CKD at baseline, a total of 1,019 adults were eligible for the current analysis of plasma fatty acid biomarkers. Among 4,363 adults from 3C Bordeaux and Montpellier study centres, 710 adults were involved in an ancillary study on erythrocyte fatty acids and among them, there was 422 adults with available data on fatty acid biomarkers and eGFR at baseline and follow-up visits, who were included in the analysis for the secondary outcomes. For primary outcomes of incident chronic kidney disease, after excluding 49 adults with prevalent CKD at baseline, a total of 373 adults were eligible for the current analysis of erythrocyte fatty acids.

Measurement of fatty acid biomarker concentrations:
In the 3C study, fasting blood samples were collected at the baseline visit into heparinised evacuated tubes and centrifuged at 1000 x g for 10 min. Total lipids were extracted from plasma with 5 mL of hexane/isopropanol (3:2, by vol). The plasma fatty acid composition was determined from 2 mL of the lipid extract after transformation into isopropyl esters. Separation of isopropyl esters was made on a gas chromatograph (Trace, Thermoelectron, France) using a 25-m Carbowax capillary column (internal diameter: 0.32 mm). Column conditions were 180 °C for 5 min, increasing by 7.5°C/min to 220°C for 30 min. The injector was set at 60 °C and the flame ionisation detector at 250°C. Helium was used as the carrier gas (flow rate: 2 mL/min). The peaks were identified by comparison with reference fatty acid esters (Sigma, St Louis, MO), and peak areas were measured with an automatic integrator (DP700; Fisons Instruments, France). The results for each fatty acid were expressed as a percentage of total fatty acids.
Erythrocyte membrane phospholipid fatty acids were measured at the French Institute for Fats and Oils (ITERG). Total lipids from red blood cell membranes were extracted as described previously. 36 A one dimensional thin-layer chromatography was used to separate total phospholipids of red blood cells from neutral lipids. Total fatty acids of the red blood cell phospholipid fraction were methylated to obtain fatty acid methyl esters. Individual fatty acid methyl esters were separated using a gas chromatograph (Focus GC, Thermo Scientific, France) equipped with a flam ionisation detector and a split injector. A fused silica capillary column (BPX 70, 60m x 0.25mm internal diameter, 0.25mm film; Phenomenex, Germany) was used with H2 as the carrier gas (inlet pressure: 1 bar). The column temperature was programmed to increase from 150 to 200°C at 1.5°C/min for 25 min, and then from 200 to 225°C at 20°C/min and was held at 225°C until the completion of the analysis (20 min). The injection port and detector were maintained at 250 and 280°C, respectively. Data were integrated using the ChromQuest Software (Thermo Scientific). Individual fatty acid methyl esters were identified by comparing their retention times with those of authentic standards eluted in the same conditions (Sigma Chemical Co., Saint Quentin Fallavier, France). The results are expressed as a weight percentage of total fatty acids.
Physical activity assessment: Regular exercise was defined as engaging in moderate to vigorous physical activity (defined as recreational walking ≥1 hours per day or practicing sport ≥1 times per week).

Statistical methods for incident CKD analysis: Cox regression
Missing data in covariates: Only body mass index and physical activity had missing data (2.3% and 16% respectively). Participants with missing data in body mass index (continuous) were excluded from the analysis. Missing values for physical activity were imputed to the highest frequency category.

Covariates
All the covariates were prespecified and harmonised between studies. These covariates include age (years); sex (male/female); race (Caucasian/non-Caucasian, or study-specific); clinical centre / field site, if applicable (studyspecific categories); education (<high school, high school graduate or >high school; if unavailable, cohort-specific categories); occupation (using cohort specific categories); body mass index (kg/m 2 ); smoking (current, former or never; if history of smoking not assessed, then current or non-current); alcohol intake (none, <1 drink/day, 1-2 drink/day or >2 drink/day; if unavailable, cohort-specific categories); physical activity (quartiles of metabolic equivalents per week; if unavailable, cohort-specific categories or definition); prevalent coronary heart disease (defined by ICD-9 410-414, or ICD-10 I20-I25, or cohort-specific definitions); use of lipid-lowering drugs; prevalent diabetes mellitus (defined as treatment with oral hypoglycaemic agents or insulin, fasting glucose ≥ 126mg/dL, or cohort-specific definitions); urine albumin-creatinine ratio (mg/g); systolic blood pressure (mm Hg); use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (yes/no); and use of other anti-hypertensive drugs (yes/no) (if data on specific types of anti-hypertensive drugs were not available, then use of any anti-hypertensive drugs).

Declined to participate due to very limited number of incident CKD case:
Health  a For statistical analyses, covariates were pre-specified and standardised across studies with harmonised definitions and categorisation: For physical activity, the first preference is quartiles of METs or continuous METs. If METs are not available, then the second preference is the use of four categories of physical or leisure activity as defined in individual studies, or cohort-specific definitions. Prevalent diabetes mellitus was defined as treatment with oral hypoglycaemic agents or insulin, fasting glucose >126 mg/dL, or cohort-specific definitions. Prevalent coronary heart disease was defined by ICD-9 410-414, or ICD-10 I20-I25, or cohort-specific definitions. Albuminuria was defined as urinary albumin-creatinine ratio ≥30 mg/g. Study. a FDPS, MAS and METSIM did not contribute to the sensitivity and the secondary analyse of ≥40% decline in eGFR due to very low numbers of incident cases (n ≤11). b For the secondary outcomes, participants with prevalent CKD at baseline were not excluded in the analysis, resulting in a larger sample size compared to the primary outcome. c The total number included the sample size for the erythrocyte phospholipid for the 3C study since this biomarker compartment was used in the overall pooled analysis. d The annual rate was calculated by dividing the percentage of cases with the median follow-up duration for each cohort. a Incident CKD was defined as an eGFR <60 mL/min/1.73 m 2 during follow-up among participants with baseline eGFR ≥60 mL/min/1.73 m 2 . Effect estimates were pooled using inverse-variance weighted meta-analysis. b The small difference in number of cases was due to missing measurement for specific n-3 PUFA fatty acids in some cohorts. c Model 1 adjusted for age, sex, race, clinical centre / field site, education, occupation, body mass index, smoking, alcohol intake, physical activity, prevalent coronary heart disease, and use of lipid-lowering drugs, where applicable. Model 2 adjusted for all covariates in Model 1 and additionally adjusted for prevalent diabetes mellitus, urine albumin-creatinine ratio, systolic blood pressure, use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and use of other anti-hypertensive drugs. d For the sum of seafood n-3 PUFA (EPA+DPA+DHA), as DPA was not available in three of the cohorts (CCCC, InCHIANTI and ULSAM), the sum in these cohorts was calculated as EPA+DHA instead. 0.0 Abbreviations: CKD, chronic kidney disease; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; eGFR, estimated glomerular filtration rate; EPA, eicosapentaenoic acid; PUFA, polyunsaturated fatty acids; RR, relative risk. a Incident CKD was defined using more stringent criteria as a follow-up eGFR <60 mL/min/1.73 m 2 and <75% of baseline eGFR among participants with baseline eGFR ≥60 mL/min/1.73 m 2 . Effect estimates were pooled using inverse-variance weighted meta-analysis. b Number of cohorts contributing to this analysis was lower than the primary outcome because three cohorts were excluded due to the very low number of incident cases (FDPS, MAS and METSIM). c The small difference in number of cases was due to missing measurement for specific n-3 PUFA fatty acids in some cohorts. d Model 1 adjusted for age, sex, race, clinical centre / field site, education, occupation, body mass index, smoking, alcohol intake, physical activity, prevalent coronary heart disease, and use of lipid-lowering drugs, where applicable. Model 2 adjusted for all covariates in Model 1 and additionally adjusted for prevalent diabetes mellitus, urine albumin-creatinine ratio, systolic blood pressure, use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and use of other anti-hypertensive drugs. e For the sum of seafood n-3 PUFA (EPA+DPA+DHA), as DPA was not available in three of the cohorts (CCCC, InCHIANTI and ULSAM), the sum in these cohorts was calculated as EPA+DHA instead. Abbreviations: CKD, chronic kidney disease; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; PUFA, polyunsaturated fatty acids. a Data are relative risk (95% CI) per interquintile range of each n-3 PUFA biomarker. Incident CKD was defined as an eGFR <60 mL/min/1.73 m 2 during follow-up among participants with baseline eGFR ≥60 mL/min/1.73 m 2 . Model 1 adjusted for age, sex, race, clinical centre / field site, education, occupation, body mass index, smoking, alcohol intake, physical activity, prevalent coronary heart disease, and use of lipid-lowering drugs, where applicable. Model 2 adjusted for all covariates in Model 1 and additionally adjusted for prevalent diabetes mellitus, urine albumin-creatinine ratio, systolic blood pressure, use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and use of other anti-hypertensive drugs. b The overall pooled analyses were performed after excluding data from one cohort at a time. c For the sum of seafood n-3 PUFA (EPA+DPA+DHA), as DPA was not available in three of the cohorts (CCCC, InCHIANTI and ULSAM), the sum in these cohorts was calculated as EPA+DHA instead. d For studies evaluating multiple biomarker compartments, results from alternative fractions (i.e., total plasma for 3C instead of erythrocyte phospholipids, and plasma phospholipids for METSIM instead of erythrocyte phospholipids) were utilised in place of results from phospholipids. ALA, α-linolenic acid; CKD, chronic kidney disease; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; RR, relative risk. a Data are relative risk (95% CI) per interquintile range of each n-3 PUFA biomarker. Incident CKD was defined as an eGFR <60 mL/min/1.73 m 2 during follow-up among participants with baseline eGFR ≥60 mL/min/1.73 m 2 . Effect estimates were pooled using inverse-variance weighted meta-analysis. P for heterogeneity across three regions was assessed by meta-regression. b For the MAS cohort, which was conducted in Australia, data were combined with other European cohorts. c Model 1 adjusted for age, sex, race, clinical centre / field site, education, occupation, body mass index, smoking, alcohol intake, physical activity, prevalent coronary heart disease, and use of lipid-lowering drugs, where applicable. Model 2 adjusted for all covariates in Model 1 and additionally adjusted for prevalent diabetes mellitus, urine albumin-creatinine ratio, systolic blood pressure, use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and use of other anti-hypertensive drugs. d For the sum of seafood n-3 PUFA (EPA+DPA+DHA), as DPA was not available in three of the cohorts (CCCC, InCHIANTI and ULSAM), the sum in these cohorts was calculated as EPA+DHA instead. ALA, α-linolenic acid; CKD, chronic kidney disease; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; RR, relative risk. a Data are relative risk (95% CI) per interquintile range of each n-3 PUFA biomarker. Incident CKD was defined as an eGFR <60 mL/min/1.73 m 2 during follow-up among participants with baseline eGFR ≥60 mL/min/1.73 m 2 . Effect estimates were pooled using inverse-variance weighted meta-analysis. P for heterogeneity across three regions was assessed by meta-regression. b Model 1 adjusted for age, sex, race, clinical centre / field site, education, occupation, body mass index, smoking, alcohol intake, physical activity, prevalent coronary heart disease, and use of lipid-lowering drugs, where applicable. Model 2 adjusted for all covariates in Model 1 and additionally adjusted for prevalent diabetes mellitus, urine albumin-creatinine ratio, systolic blood pressure, use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and use of other anti-hypertensive drugs. c For the sum of seafood n-3 PUFA (EPA+DPA+DHA), as DPA was not available in three of the cohorts (CCCC, InCHIANTI and ULSAM), the sum in these cohorts was calculated as EPA+DHA instead.